Asparagine-linked oligosaccharides of BHK cells treated with inhibitors of oligosaccharide processing

• Published in: Biochemical journal Vol. 247; no. 3; pp. 537 - 545
• Main Authors: Hughes, R C, Foddy, L, Bause, E
• Format: Journal Article
• Language: English
• Published: England 01.11.1987
• Subjects: 1-Deoxynojirimycin; alpha-Mannosidase; Animals; Asparagine; asparagine-linked; Cell Line; Chromatography, Affinity; Chromatography, Gel; Cricetinae; Glucosamine - analogs & derivatives; Glucosamine - pharmacology; Glucose - pharmacology; Glycopeptides - analysis; Glycosylation; hamsters; kidney; Lectins - pharmacology; Mannose - metabolism; Mannosidases - pharmacology; Oligosaccharides - antagonists & inhibitors; Oligosaccharides - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2470537

Baby-hamster kidney (BHK) cells were labelled with [2-3H]mannose for 1-2 days in media containing 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin or 1-deoxymannojirimycin. Glycopeptides obtained by Pronase digestion of disrupted cells were analysed by lectin affinity chromatography, by Bio-Gel P4 gel filtration and by paper chromatography of oligosaccharides released by endo-beta-N-acetylglucosaminidase H. Biosynthesis of complex-type oligosaccharides was diminished but not abolished, the greatest effect being obtained by continuous culture of cells with 1-deoxymannojirimycin. Under these conditions cells contained only 20-30% of the concentration of complex-type chains found in control cells and correspondingly increased amounts of oligomannose-type chains. Similar concentrations of asparagine-linked Man6-GlcNAc2 and Man5GlcNAc2 were present in 1-deoxymannojirimycin-treated cells and control cells, indicating that the inhibition of complex-type chain formation was not related simply to an inability of inhibitor-treated cells to carry out extensive mannosidase-catalysed processing. N-Methyl-1-deoxynojirimycin induced accumulation of oligomannose-type chains containing three glucose residues, and cells treated with 1-deoxynojirimycin contained oligosaccharides with one to three glucose residues. Cells cultured in the presence of the inhibitors retained sensitivity towards the galactose-binding lectins ricin and modeccin.